Monocytes are generated in the bone marrow (BM) to be released in the blood stream and give rise to different types of tissue-macrophages ordendritic cells after leaving the circulation. Monocytes, their progeny and immediate precursors in the bone marrow have also been named the ‘mono-nuclear phagocyte system’ (MPS). They are derived from granulocyte/macrophage colony forming unit (CFU-GM) progenitors in the bone marrow that gives rise to monocytic and granulocytic cells. The maturation process of the monocytic lineage in vivo passes from a monoblast stage, through the promonocyte stage to mature monocytes (Goud T J et al. 1975). IL-3, GM-CSF and macrophage colony stimulating factor (M-CSF) stimulate in vivo generation of monocytes (Metcalf D et al. 1990). In vitro, hematopoietic progenitor cells cultured with GM-CSF induce CFU-GM to differentiate towards granulocytes, while addition of FL and SCF shifts differentiation from the granulocytic to the monocytic lineage (Gabbianelli M et al. 1995; Willems R et al. 2001).
Several recent studies indicate that although specific surface marker expression may vary in detail between species, the general differentiation pathways of monocytes and their progeny appear to be largely conserved between mice and humans (Gordon S et al. 2005). With the higher accessibility to experimentation a more detailed differentiation pathway could be worked out in the animal model. Mouse monocytes originate from hematopoietic stem cells (HSC) in the bone marrow via successive commitment steps and several intermediate prognitor stages with increasingly restricted differentiation potential (Shizuru J A et al. 2005; Kondo M et al. 2000). This differentiation series is believed to pass through the common myeloid progenitor (CMP), which can give rise to all myeloid cells, to the granulocyte-macrophage progenitor (GMP), which gives rise to monocytic and granulocytic cells and may be identical or very similar to CFU-GM progenitors. Yet a more immediate monocytic progenitor in the bone marrow appears to be the macrophage/dendritic cell progenitor (MDP) that can give rise to macrophages and dendritic cells, likely via a monocyte intermediate stage (Fogg et al 2006).
Newly formed monocytes leave the BM within 24 hours and migrate to the peripheral blood. Circulating monocytes can adhere to endothelial cells of the capillary vessels and are able to migrate into various tissues (van Furth R. et al. 1992), where they can differentiate into macrophages or dendritic cells. These adherence and migration involve surface proteins, lymphocyte-function associated antigen-1 (LFA-1), CD11 and antigen-4 (VLA-4), belonging to the integrin superfamily of adhesion molecules (Kishimoto T K et al. 1989). These integrins interact with selectins on endothelial cells. Monocyte derived macrophages can show a high degree of heterogeneity that reflects a morphological and functional specification adopted in the infiltrated tissue. According to their anatomical localization they may also have distinct names (e.g. microglia in the central nervous system and Kupffer cells in the liver).
Although it remains controversial, whether some resident macrophage populations may be capable of proliferating in situ under certain conditions, the majority of macrophages appear to have no or a very limited proliferation capacity (Gordon S, et al., 2005). The renewal of tissue macrophage populations therefore depends on the influx of monocytes and their local differentiation (Crofton R W et al. 1978; Blusse van Oud Alblas A et al; 1981). Although such tissue infiltrating monocytes can have a very limited proliferation ability, monocytes circulating in the blood do not cycle and rapidly differentiate into macrophages rather than expand, when stimulated with M-CSF ex vivo.
Monocyte recruitment to tissues differs under homeostatic and inflammatory conditions and appears to involve two distinct monocyte populations that have been identified in humans and mammalian animal models (Gordon S, et al. 2005). During inflammation monocytopoiesis increases (Shum D T et al. 1982; van Waarde D et al. 1977) resulting in elevated monocyte numbers. Furthermore, inflammatory mediators, IL-1, IL-4, IFN-γ and TNF-α upregulate expression of selectins on endothelial cells, promoting migration of monocytes into tissues. The same cytokines modulate expression of integrin adhesion molecules on monocytes (Pober J S et al. and 1990). At the site of inflammation monocytes are involved in the phagocytosis of opsonized microorganisms or immune complexes via surface γ receptors (CD64, CD32) and complement receptors (CD11b, CD11c). The microorganisms are synergistically killed by reactive oxygen and nitrogen metabolites and through several hydrolytic enzymes (acid phosphatase, esterase, lysozyme and galactosidase) (Kuijpers T. 1989; Hibbs J B et al. 1987). Importantly, monocyte derived macrophages and dendritic cells stimulate T cells by antigen presentation and thus, are involved in the recognition and activation phases of adaptive immune responses (Nathan C F. 1987). Monocytes also secrete a large number of bioactive products which play an important role in inflammatory, proliferative and immune responses, including growth factors (GM-CSF, G-CSF, M-CSF, IL-1) and antiproliferating factors (IFNs, TNF).
Lipopolysaccharide (LPS) or endotoxin is a predominant integral structural component of the outer membrane of Gram-negative bacteria and one of the most potent microbial initiators of inflammation. LPS binds to the CD14 glycoprotein that is expressed on the surface of monocytes and stimulates the toll receptor pathway via activation of TLR4. Other PAMPs (pathogen associated molecular patterns) can also initiate inflammatory responses via other TLR receptors. The binding of LPS or other PAMPS induces production of TNF-α, IL-1, -6, -8 and -10 (Wright S D. Et al; 1990; Dobrovolskaia M A et al. 2002; Foey A D. et al. 2000).
Other than LPS or other PAMPs, one of the most efficient stimuli for cytokine production in vitro is the direct cell-cell contact of monocytes with activated lymphocytes (Wey E. et al. 1992; Parry S L. Et al. 1997), via CD40 ligand (CD40L) (Wagner D H. Et al. 1997; Shu U. et al. 1995; Alderson M R. et al. 1993). This interaction may also be important in the immune surveillance of tumors. Thus the incubation of monocytes with CD40L-transfected cells results in tumoricidal activity against a human melanoma cell line. Furthermore functional interactions have also been described between monocytes and NK cells, a cell type with significant anti-tumor activity. Both direct cell-cell contact (Miller J S. Et al. 1992). and release of soluble factors such as IL-12, TNF-α, IL-15 or IL-1β by activated monocytes induce proliferation, production of IFN-γ (Carson W E et al. 1995; Tripp C S. et al. 1993) and the cytotoxic potential of cocultured NK cells in a time dependent manner (Chang Z L. et al. 1990; Bloom E T. et al. 1986).
Finally macrophages are also critically involved in woundhealing and tissue repair, where they assume trophic functions by removing debris and orchestrating the recruitment and activity of other cell types participating in tissue remodelling (Gordon S et al. 2003)
Dendritic cells (DCs) are components of the innate immune system. They are antigen presenting cells with the unique ability to induce a primary immune response (Banchereu et al. 2000). They can be derived from circulating monocytes or circulating DC progenitors in the blood and non-lymphoid peripheral tissues, where they can become resident cells (Bancherreau J. et al. 1998, 2000) (Geissmann, 2007) (Wu and Liu, 2007). Immature DCs (iDCs) recognize pathogens through cell surface receptors, including Toll-like receptors (Reis e Sousa C. 2001). After uptake of antigen DCs mature and migrate to lymph nodes. Mature DCs (mDCs) are efficient antigen presenting cells (APCs) which mediate T cell priming (Banchereau J. et al. 1998, 2000). Furthermore a predominant role of DCs has been described in NK cell activation in mice and humans. Both immature and bacterially activated human monocyte-derived DCs have been shown to induce cytokine secretion and cytotoxicity by NK cells (Ferlazzo G. et al. 2002; Fernandez N C. et al. 1999).
The in vitro differentiation of murine macrophages from bone marrow in the presence of M-CSF was described by Stanley et al. (1978, 1986). Whereas progenitor cells will initially proliferate in response to M-CSF, they eventually differentiate to mature macrophages and terminally withdraw from the cell cycle (Pixley and Stanley, 2004). Thus even though the macrophages generated this way will survive for a limited time, they are not homogeneous and cannot be further expanded in culture. Similarly human monocytes do not proliferate in response to M-CSF but initiate morphological changes indicative of macrophage differentiation (Becker et al., 1987). Although a significant number of monocytes can be obtained from a patient by leukapheresis and elutriation (Stevenson et al., 1983), these cells will further differentiate to macrophages in a few days without proliferating and cannot be maintained in culture.
Now, the present invention provides a new in vitro method for generating, maintaining and expanding monocytes and macrophages in long term culture.
The inventors have indeed demonstrated that it is possible to expand and maintain monocytes and macrophages in culture for weeks or months, by inactivating the expression of MafB and c-Maf in said cells. Not only in vitro generated macrophages but also mature bone marrow MafB and c-Maf deficient macrophages and blood monocytes continue to proliferate in culture.
Methods of the invention may thus be useful for therapeutic approaches requiring amplification of monocytes and monocyte derived cells, as well as for screening for drugs targeting monocyte, monocyte derived macrophages (including osteoclasts) and dendritic cells or for testing the response to specific drugs in a patient specific way, or for studying the molecular basis of monocyte or monocyte derived cell dependent diseases by culturing and expanding monocyte or monocyte derived cells of afflicted patients.